APPLICATION OF CONFOCAL LASER-SCANNING MICROSCOPY TO ANALYSIS OF H2O2- INDUCED DNA-DAMAGE IN HUMAN-CELLS

Confocal laser scanning microscopy (CLSM) offers improved depth discrimination and spatial resolution to the analysis of biologic samples. We demonstrate in this paper that such technology is valuable in examining DNA single-strand breaks in human cells. The single-cell-gel (SCG) assay is a new technique for measuring DNA strand breaks in individual cells. Cells embedded in low-melting-point agarose are treated with varying concentrations of hydrogen peroxide to induce DNA strand breaks. Following cell lysis and alkaline electrophoresis, which enables single-stranded break detection, analysis of the resulting ''comets'' provides an accurate method of comparing changes in DNA migration patterns, which have been shown to reflect the DNA damage levels. A statistically significant difference (p < 0.01) in single-stranded DNA damage levels was detected in cells exposed to hydrogen peroxide concentrations as low as 10 nm for 2 min. LSM analysis of the SCG technique allows rapid, sensitive, and reproducible quantitation of single-stranded breaks of cellular DNA.